Preparation and characterization of PEG-modified PCL nanoparticles for oxygen carrier: a new application of Fourier transform infrared spectroscopy for quantitative analysis of the hemoglobin in nanoparticles

Figures & data

Table I. The five ratio-points of calibration standards weighed carefully for the four linear regression curves.^a,b,c

Samples	Ratios of Hb: BPN:PAN	Actual weights of each components (g)
		Hb	PAN	BPN	Total weight (g)
PCL	1:0.3:6	0.2739	1.6440	0.0820	1.9999
	1.25:0.3:6	0.3310	1.5893	0.0800	2.0003
	1.5:0.3:6	0.3847	1.5386	0.0769	2.0002
	3:0.3:6	0.6453	1.2905	0.0643	2.0001
	6:0.3:6	0.9757	0.9756	0.0489	2.0002
mPEG-PCL [CL]/[EG] = 9:1	1:0.3:6	0.2742	1.6440	0.0822	2.0004
	1.25:0.3:6	0.3313	1.5894	0.0794	2.0001
	1.5:0.3:6	0.3848	1.5387	0.0765	2.0000
	3:0.3:6	0.6450	1.2903	0.0645	1.9998
	6:0.3:6	0.9757	0.9756	0.0489	2.0002
mPEG-PCL [CL]/[EG] = 7:3	1:0.3:6	0.2741	1.6436	0.0823	2.0000
	1.25:0.3:6	0.3313	1.5894	0.0795	2.0002
	1.5:0.3:6	0.3849	1.5382	0.0771	2.0002
	3:0.3:6	0.6452	1.2903	0.0644	1.9999
	6:0.3:6	0.9756	0.9754	0.0488	1.9998

^aThe calibration standard samples (Sample 1–5) were mixture of Hb-BPN-PAN according to the five levels of weight ratio(w/w/w). ^bBiodegradable BPN were formulated by PCL and mPEG-PCL (PEG mole fraction of 10% and 30%, respectively), and all weighed according to the same ratio above. ^cInternal standard PAN used for calibration curves establishment was added into the mixture for a constant weight.

Table II. The actual weights of nanoparticles and PAN of each sample with the ratio be around 1:4.

Samples^a	HbPN0	HbPN10	HbPN30
WNP(g)	0.0151 ± 0.0001	0.0149 ± 0.0002	0.0151 ± 0.0002
WPAN(g)^b	0.0599 ± 0.0002	0.0601 ± 0.0002	0.0600 ± 0.0001

^aThe samples HbPN0 to HbPN30 were prepared by biodegradable shell polymers: PCL and mPEG-PCL (PEG mole fraction of 10 and 30%, respectively), using the identical preparation process. ^bInternal standard PAN used for quantitation analysis was added into the mixture for a constant weight.

Figure 1. TEM image of mPEG-PCL with PEG mole ratio of 30% (HbPN30) revealing shape and sizes of nanoparticles encapsulating hemoglobin molecules.

Table III. Particles size and surface charge of hemoglobin-loaded polymeric nanoparticles encapsulated by four types of polymer.

Samples^a	PEG content in copolymer (%)	Mean diameter (nm)	Polydispersity index	Contact angle (°)	ζ potential (mV)
HbPN0	0	180.0 ± 13.6	0.196	120.5 ± 0.7	−31.3 ± 0.3
HbPN10	10	187.8 ± 10.3	0.213	75.3 ± 1.2	−25.8 ± 0.7
HbPN30	30	171.1 ± 16.5	0.193	30.9 ± 1.8	−18.4 ± 0.8

^aThe samples HbPN0 to HbPN30 were prepared by the degradable shell polymers: PCL and mPEG-PCL (PEG mole fraction of 10 and 30%, respectively) by the identical preparation process.

Figure 2. FTIR spectra of PAN, Hb and the polymers used as nanoparticles shells. (a) The special absorbance peaks in FTIR were (1) the bending vibration of N–H (amide II) at 1540 cm⁻¹ attributed to Hb and (2) the stretching vibration of –C≡N at 2241 cm⁻¹ attributed to PAN, respectively; (b) the bands (1) Hb and (2) PAN used in this quantification method had no interference signals with the polymers used in experiments.

Figure 3. Infrared spectra of Span-80 and Tween-80 comparing to blank nanoparticles encapsulated by Span-80 and Tween-80. There were no interference peak from Span-80 and Tween-80 at PAN special absorbance peaks 2241 cm⁻¹. Furthermore, note that the disappearance of the Span-80 absorbance peak at Hb special absorbance peaks 1560 cm⁻¹ after nanoparticles formed which could verify the specificity of this promoted method.

Figure 4. Regression lines of polymeric Hb-BPN-PAN between the hamide II/hPAN ratio (y) and the weight ratio of Hb to PAN (x). The diblock polymeric nanoparticles were fabricated by PCL, mPEG-PCL with PEG mole fraction of 10% and mPEG-PCL with PEG mole fraction of 30% in the identical preparation process.

Table IV. Accuracy (percent recovery) of the method for the determination of Hb content in Hb-BPN mixture (n = 5).

Samples^a concentration		Theoretical hemoglobin amount (g)	Measured hemoglobin amount (g)	Analytical recovery (%)	Average recovery ± relative standard deviation (%)
HbPN0	Low	0.0105	0.0103	98.09524	100.56 ± 2.14
	Medium	0.0155	0.0158	101.9355
	High	0.0303	0.0308	101.6502
HbPN10	Low	0.0105	0.0104	99.04762	97.18 ± 1.71
	Medium	0.0155	0.015	96.77419
	High	0.0303	0.029	95.70957
HbPN30	Low	0.0103	0.0101	98.05825	98.60 ± 0.93
	Medium	0.0155	0.0152	98.06452
	High	0.0301	0.03	99.66777

^aThe samples HbPN0 to HbPN30 were prepared by the degradable shell polymers: PCL and mPEG-PCL (PEG mole fraction of 10 and 30%, respectively) by the identical preparation process.

Table V. The intra- and inter-day precision (relative standard deviation) of the method for the determination of Hb content in Hb-BPN mixture (percent of drug loading).

Hemoglobin amount in mixture of Hb-BPN-PAN^a (g)		Intra-day (n = 5) (< 5%) precision	Inter-day (n = 4) (< 15%) precision
HbPN0	0.0201 ± 0.0002	2.17	6.41
	0.0301 ± 0.0001	1.87	3.63
	0.0401 ± 0.0001	1.58	5.63
HbPN10	0.0202 ± 0.0002	2.31	4.57
	0.0301 ± 0.0003	1.58	3.82
	0.0401 ± 0.0001	2.71	3.46
HbPN30	0.0200 ± 0.0002	2.23	4.77
	0.0298 ± 0.0002	1.88	5.61
	0.0401 ± 0.0001	1.75	3.81

^aThe samples HbPN0 to HbPN30 were prepared by the degradable shell polymers: PCL and mPEG-PCL (PEG mole fraction of 10 and 30%, respectively) by the identical preparation process.

Figure 5. Drug loading and relative standard deviation in HbPN. The samples HbPN0 was prepared by PCL. The samples HbPN10 to HbPN30 were prepared by copolymer mPEG-PCL and the PEG percentage in copolymer were 10 and 30%.

Figure 6. Schematic representation of the relationship between some Hb liquid droplet and low or high density. PEG brush structure on the surface of nanoparticles. (a) The PEG chains with low density would form entrance for hemoglobin being incorporated into inner water phase during the formation of HbPN. (b) When more density of PEG segments was surrounding the nanoparticles, the stronger steric repulsion was produced to prevent not only from opsonins adsorption but Hb encapsulated into inner phase.